Simulations of neural synapses overturn some longstanding assumptions

Each time Sergei Rachmaninoff sat down to perform his
virtuosic Piano Concerto No. 3, he played more than 28,000 notes
in about 30 minutes, his fingers, a blur of muscular energy,
striking the keys with the precise force — at the precise
instant — the maestro intended. With only faint
electrochemical signals running through nerves of greatly varied
length, how is it possible for the brain to initiate and control
such rapid-fire movement, dozens of muscles simultaneously, with
exquisite sensitivity and split-second timing?

Over the past century, scientists have taken many steps
toward understanding the biology underlying such feats. The
synapse — where nerve cells meet or connect to a muscle
fiber — amplifies the tiny voltage generated in the brain
into forces large enough to pound out a thunderous finale.
Still, these intricately coordinated mind-body processes hold
many mysteries.

PSC senior scientist Joel Stiles and his collaborators solved
a few recently by using a supercomputing system to recreate the
business end of a synapse. Their tools included MCell, powerful
software — co-authored by Stiles and Thomas Bartol of the
Salk Institute — for simulating the microphysiology of
interacting cells, coupled with DReAMM visualization software
developed in Stiles’ lab at PSC. The computational work
relied heavily on Jonas, PSC’s 128-processor shared-memory
HP system dedicated to biomedical research. Stiles and
colleagues modeled two kinds of synapses in unprecedented
detail, and their findings, published recently in Science and
other journals, overturn some longstanding assumptions about
neural communication, offer insight into a family of crippling
diseases, and demonstrate the power of computation allied with
experimental measurement.

A Barrage of Neurotransmitters

The secret to coordination, says Stiles, is predictability.
As a child grows, the motor cortex in the brain develops
increasingly sophisticated programs of voluntary muscle control.
These programs must anticipate delays that occur as a command
impulse travels to the spinal cord, out a peripheral nerve, and
across the nanometers-wide synaptic cleft that separates a
neuron from the muscle fiber it innervates.

Decades ago, biologists measured the signal delays introduced
by nerve-muscle synapses, and found that the lag time is
amazingly consistent, varying less than 30 millionths of a
second from one synaptic firing to another. How does the body
manage this feat of consistency?

Over the years, molecular biologists have filled-in much of
the story. On its way from brain to limb, an impulse shoots down
the nerve cell to its terminus and arrives in the form of a
rapid change in sodium ion concentration. That creates a
voltage. The voltage triggers thousands of sphincter-like
proteins embedded in the cell wall of the neuron; these protein
channels open, and some allow calcium ions to flow into the
cell. As the calcium ions enter, they diffuse throughout the
interior of the neuron, bumping into tiny spherical containers
— called vesicles — arranged in neat rows close to
the interior wall.

Loaded with the neurotransmitter acetylcholine, the vesicles
are like a battery of fireworks poised to launch a
neurotransmitter barrage into the synaptic cleft — where
this amplified signal will in turn energize the adjacent muscle
fiber. In the last step, what biologists call a “fusion
event,” the calcium ions trigger some of the vesicles to
fuse with the cell membrane and open outward, allowing their
payload to escape.

The details of this critical last step, however, have so far
stumped neuroscientists. “We still don’t know how
calcium binds to receptor areas on the vesicles to lead to this
fusion event,” says Stiles. “Nor do we know how many
protein channels need to open to admit the calcium, or how far
the calcium can diffuse to find the vesicles and cause them to
fuse. Does a calcium channel have to open right next to a
vesicle, or can the ions come in through a variety of different
gates and collectively trigger a variety of vesicles?”

A New Picture of Fusion

To help answer these questions, Stiles teamed up with PSC
researcher John M. Pattillo (now at Macon State College) and
neurobiologist
Stephen D. Meriney of the University of Pittsburgh. With
support from the National Institutes of Health, the group was
able to take a hybrid approach that combined classic empirical
observation with a sophisticated Monte Carlo supercomputer
simulation.

Two Active Zone Architectures

Cutaway views into the interior of a nerve cell show low
(top) and high magnification views of two different active
zone configurations simulated with MCell by Stiles and
colleagues. The simulations include diffusing calcium ions
(small blue dots) and synaptic vesicles (large purplish
spheres) with calcium binding sites (red & yellow) and
calcium channels (color-coded glyphs in blue triangles) in
the nerve membrane. This model has a small number of binding
sites on each vesicle. Translucent boxes are used to count
calcium ions in different regions of space. (DReAMM image by
Joel Stiles, PSC).

Stiles and Pattillo used computer-aided design tools to
create a three-dimensional model of an entire “active
zone,” the region inside the neuron where dense arrays of
vesicles dock and fuse. Then, using MCell they created a
simulation that tracks each calcium channel, the calcium binding
sites on vesicles, and thousands of diffusing calcium ions
inside the micron-wide active zone for several milliseconds of
the firing cycle.

In the laboratory, Meriney probed living nerve and muscle
cells to record how calcium concentration spikes and then falls
following an electrical impulse. “These and other
experimental results constrain many of the variables,”
explains Stiles, “leaving us with only a few free
parameters to play with in the simulations.” Running the
simulations over and over, Stiles and Pattillo looked for
combinations that would reproduce the recorded behaviors of real
synapses: for example, the short and consistent delay between
the stimulating impulse and neurotransmitter release.

Long used in high-energy physics, astronomy and other areas
of science, such a hybrid approach to modeling, notes Stiles,
has been less used in biology. “In part, this is because
biology is so complicated and difficult to measure on these
scales, and in part because the computational cost is so high.
We are only now getting to the point where we have the
supercomputer power and the insight into biomolecular dynamics
to do computational biology this way.”

To thoroughly explore the plausible range of permutations,
Stiles and Pattillo had to run roughly 500,000 simulations. Each
run generated thousands of output files, so the group devised a
compression scheme, analogous to the MPEG encoding used for DVD
movies, that allowed them to efficiently store the results and
mine them for insights.

After more than a year of patient work, the data mining
struck gold — in a surprising place. Neuroscientists had
for years guessed that each synaptic vesicle sports four binding
sites for calcium ions, and that fusion occurs only when ions
dock at all four. This was one of the first models Stiles and
Pattillo tried. “The results made it immediately
obvious,” recalls Stiles, “that this wasn’t
right.” The virtual neuron almost never released
transmitter, and no amount of tweaking other variables could
produce realistic behavior. “We scratched our head and
said, ‘OK, let’s push up the number of binding sites
on each vesicle and see what happens.’”

After testing many different combinations, the group finally
discovered one that neatly reproduces the experimental data. In
this model, each vesicle is dotted with 25 to 40 binding sites,
and fusion occurs when calcium ions fill six to eight of those
sites. “The latest data coming in from biochemists now
suggest that there are good reasons to expect this is
true,” says Stiles. “So that is quite
gratifying.”

MCell simulations have proven ability at revealing the details
of cell-to-cell interactions

Neurotransmission in the Ciliary Ganglion

A nerve from the brain leads to the ciliary ganglion,
which controls the iris and lens of the eye. This
visualization from an MCell simulation represents
neurotransmitter molecules (small green ovoids) diffusing
from vesicles (translucent yellow spheres) in a
reconstructed ciliary ganglion synapse. Different
neurotransmitter receptors (small red circles & blue
squares) are also represented. This simulation showed that
neurotransmitters release at ectopic (non-active zone) sites
as well as from active zones. (DReAMM image by Thomas
Bartol, Salk Institute).

The achievement builds on another project to which Stiles
also contributed, in collaboration with Terrence J. Sejnowski,
Thomas M. Bartol and others at the Salk Institute and the University of California, San
Diego. That effort similarly constructed a 3-D model of a
synapse, in this case one that connects two neurons. Using a
model derived from microscope cross-sections of actual synapses,
the simulations overturned the conventional view that vesicles
release neurotransmitters only within the active zone. Fusion
events, they concluded, must be occurring in other regions of
the synapse as well.

Such detailed insights into the structure of synapses are
especially relevant for a class of diseases, called myasthenias,
that arise when synapses are malformed or attacked by the immune
system, leading to weakness, motor dysfunction, even paralysis.
Because there’s a need, and because MCell has proven
abilities, prospects are promising for this way of understanding
cell-to-cell interactions. With a soon-to-be-released new
version of MCell and DReAMM, even more precise answers will be
possible. “In the new version, molecules are able to react
chemically with each other,” says Stiles, “as they
diffuse through space. So much more general phenomena now become
potential subjects for MCell simulations.”